Hemoglobin-based blood substitutes catalyze a number of oxidation-reduction reactions which can lead to both the destruction and formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In order to assess the contribution of these reactions to the cellular toxicity of blood substitutes, we have developed in vitro models to elucidate the molecular mechanisms by which blood substitutes enhance or inhibit vascular endothelial cell damage by ROS and RNS. We have developed an in vitro model system for assessing hemoglobin-induced damage of bovine aorta endothelial cells (BAEC), the role of reactive oxygen species as mediators of damage, and the relative contribution of necrotic and apoptotic cell death pathways. Using this system we demonstrated that the pseudo-peroxidase activity of hemoglobin protected against hydrogen peroxide induced necrosis. Cross-linked hemoglobin derivatives were shown to undergo oxidative damage and to undergo a transition to a stable, ferryl (Fe+4) state in the presence of hydrogen peroxide. The isolated ferryl-hemoglobin derivatives were toxic to BAECs and induced apoptotic cell death. We also examined the interaction of bacterial lipopolysaccharide (LPS) with hemoglobin and the enhanced toxicity to endothelial cells resulting from the LPS/hemoglobin complex. Hemoglobin had no effect on apoptosis of BAEC or on CD 54 (an endothelial cell adherence protein) expression induced by LPS interaction with the high-affinity LPS receptor. Enhanced toxicity was only observed at relatively high concentrations of LPS in the absence of high-affinity LPS receptor interactions. Anti-oxidants selectively protected against enhanced toxicity of the complex, suggesting that the enhanced toxicity is primarily due to LPS induced changes in the oxidation of hemoglobin. Recent studies have implicated peroxynitrite, a highly reactive species produced by activated inflammatory cells, as a mediator of endothelial cell damage. Preliminary studies suggest that peroxynitrite induced toxicity towards BAEC is partially dependent on the oxidation of serum components. In this system, hemoglobin selectively protected against the formation of toxic serum components upon the addition of peroxynitrite. Future studies will address the serum components undergoing oxidation and the mechanism by which hemoglobin protects.